The present invention relates to a method for synthesis of sulfhydryl-containing peptides which comprises forming a benzylthioether-linked solid support-bound thiol compound, coupling at least one amino acid or peptide to said compound until a peptide of desired amino acid sequence is obtained, and cleaving the benzylthioether linkage to release the synthesized peptide from the support with concomitant regeneration of the sulfhydryl group in the peptide. The invention also relates to novel solid support-bound thiol compounds and their use for introducing sulfhydryl groups into peptides, particularly antigens and pharmaceutical peptides.
Several methods are known wherein peptides are synthesized in vitro. The principal methodology used for peptide synthesis involves variations of the solid-phase methodology developed by Merrifield and coworkers; see, for example, Erickson and Merrifield, "The Proteins", Third Edit., Vol. 2, Academic Press, New York, Chapter 3, (1976). Solid phase peptide synthesis involves attachment of a first amino acid to a solid support, such as a resin, followed by sequential addition of subsequent amino acids which results in assembly of the peptide chain on the solid support.
Peptides can also be synthesized by related methods involving coupling peptide fragments to solid supports as discussed by Erickson et al., suora, pp. 268-269. This technique involves the synthesis of small peptide segments containing a few amino acids, which segments are then coupled to each other using fragment condensation techniques to form larger peptides. Fragment condensation techniques can be combined with standard solid phase techniques wherein small peptides are attached to resins followed by sequential attachment of single amino acids or other peptide segments. Alternatively, sequential attachment of small peptides to single resin-bound amino acids can also be accomplished. The combination of the two approaches provides flexibility to synthetic schemes.
Upon completion of a particular synthesis, the synthesized peptide is then removed from the resin, usually by chemical means such as treatment with hydrofluoric acid (HF). The chemical treatment also removes various amino acid and peptide protecting groups, such as CBZ, t-Boc or Tosyl, which mask the reactivity of amino acid functional groups during synthesis.
In most peptide syntheses, the initial attachment to the resin involves the C-terminal amino acid of the peptide to be synthesized, which amino acid is covalently attached to the resin through an ester or amide linkage involving its .alpha.-carboxyl group. Synthesis then proceeds from C- to N-terminal. N-terminal to C-terminal peptide synthesis is less frequently used because the chemistry is more difficult and unwanted side reactions are more common.
The first amino acid may be covalently attached to the resin, in some cases, through its functional side chain. Initial attachment of an amino acid to the resin by means of the side chain functional group allows the possibility of bidirectional synthesis starting with the attached amino acid. Bidirectional synthesis cannot be performed if the initial amino acid is attached through the .alpha.-COOH or .alpha.-NH.sub.2 group. Side chain functional groups which have been used for attachment to resins include the sulfhydryl group of cysteine, the imidazole group of histidine, the .delta.-amino group of ornithine, the .epsilon.-amino group of lysine and the .gamma.-carboxyl group of glutamic acid. A review of the chemistry of solid phase peptide synthesis, including attachment of amino acids to resins via the .alpha.-COOH, .alpha.-NH and functional side chain groups, is found in Erickson et al., supra.
One particular example of an amino acid attachment via a functional side chain is the S-dinitrophenylene bridge between the side chain of cysteine and the support resin described by Glass et al., J. Amer. Chem. Soc. Vol. 96, pp. 6476-6480 (1974). This bridge involves a thioether linkage between the peptide and the resin which was stable to acidolysis but cleavable by thiolysis. Resin-bound cysteine, attached via the S-dinitrophenylene bridge has been used for bidirectional solid phase peptide synthesis wherein the peptide chains were extended from the attached cysteine toward both the N- and C-termini of the synthesized peptide. The peptides, therefore, had an internal cysteine.
The thioether S-dinitrophenylene bridge, however, has not been extensively used in peptide synthesis because it undergoes undesirable side reactions in the presence of base. Chemical steps commonly used in solid phase peptide synthesis cannot be used with the S-dinitrophenylene thioether-linked cysteine. Extensive reagent modification and great care are needed to minimize adverse side reactions.
Benzyl and substituted benzyl groups are known as removable protection groups which mask the reactivity of the sulfhydryl group of cysteine during peptide synthesis. An example of such use is provided in U.S. Pat. No. 3,743,628 which describes the synthesis of t-Butyloxycarbonyl-S-benzyl-L-cysteine. These protecting groups are removable by acidolysis in anhydrous HF. As discussed in Erickson et al., sucra, electron donating groups, such as methyl or methoxyl moieties, in the ortho and para positions of the benzyl group are known to increase the rate of HF cleavage. On the other hand, electron withdrawing groups in the ortho or para positions are known to decrease HF sensitivity. Other methods of cleaving S-benzyl and substituted S-benzyl groups, such as sodium in anhydrous ammonia or hydrogenolysis in anhydrous ammonia are known, but such methods are unsuitable for removing peptides bound to resins.
The introduction of thiol groups into biologically and pharmaceutically important peptides can have significant impact on the specific properties and activity of such compounds. Several naturally occurring semi-rigid cyclic peptides are known in nature. However, the majority of biologically active peptides, such as peptide hormones and neurotransmitters, are linear and flexible. In some instances conformational flexibility of such peptides gives rise to an observed lack of receptor binding specificity. Decreasing the conformational flexibility of peptide hormones and neurotransmitters provides a means to enhance biological and pharmacologic properties, such as increased receptor binding capacity and specificity.
Schiller et al., Proceedings of the Eighth American Peptide Symposium, pp. 269-278, Pierce Chemicals, 1983, discussed various means to obtain peptides with conformational restrictions. One approach is the synthesis of peptide analogs containing cyclic structures. The incorporation of sulfhydryl groups into peptides allows cyclization of small regions of the peptide through disulfide bonds produced by oxidation of the free sulfhydryl groups. In addition, thioether bonds formed by reacting a sulfhydryl group with an active akylating group introduced into an amino acid side chain may also be used to generate a cyclic structure. Peptides containing loop structures have decreased conformational freedom when compared to the corresponding linear analog.
As discussed above, such a change in conformational freedom may also be associated with a change in reactivity of the peptide. Synthesis of peptide analogs containing an intramolecular loop may also alter the susceptibility of the peptide to degradation by proteolysis. For example, peptide bonds which are substrates for proteases when present in a linear peptide sequence may become poor substrates for the same proteases when the peptide bonds lie within a small disulfide loop.
The effects of the introduction of such cyclic structures on conformational rigidity and resistance to proteolysis, therefore, can provide desirable biologic and pharmacologic properties to cyclic peptide derivatives as compared to acyclic peptides. Disulfide loops may thus be introduced into pharmaceutically useful analogs of biologically active peptides which, in their native state, do not contain such loops. (See, for example, Schiller et al., supra).
The introduction of thiol compounds into peptide sequences also provides a convenient chemical handle for further manipulation of the sulfhydryl-containing peptide. Because of their chemical reactivity such sulfhydryl groups can provide a means of covalently linking a peptide to other molecules or peptides. For example, a technique for producing antibodies to a small synthetic peptide antigen, which ordinarily would not elicit antibody production, is to conjugate the peptide to a large carrier protein such as keyhole limpet hemocyanin or bovine serum albumin. The conjugate is then used to elicit antibodies in a suitable host. One method of preparing such immunogenic conjugates is to synthesize a peptide containing a cysteine at one end. When the cysteine-containing peptide is mixed with a hemocyanin derivative containing maleimide groups, the peptide becomes covalently attached through the cysteine sulfhydryl group to the carrier protein.
According to the present invention, a new method has been found which allows the synthesis of peptides containing useful sulfhydryl groups. The method makes use of novel reagents in which thiol compounds have been attached to solid supports through a cleavable benzylthioether linkage.